| ObjectiveTo investigate the bony inductive effect of adipose drived mesenchymal stem cells(ADMSCs) differentiated into osteoblasts by the dipsacus saponin Ⅵ. To explore the bony repaired function of artificial bone graft composed of the induced osteoblasts combined with nano-hydroxyapatite / chitosan / tussah silk fibroin(n-HA / CH / TSF) for the radial bone defects in rats.MethodsRat ADMSCs were Isolated by enzymatic digestion method and purified. The surface antigens, CD31, CD44, CD49 d, CD106 were detected of 3rd passage of ADMSCs,the proliferative activity of ADMSCs was determined by MTT assay methods.The morphology change of 3rd passage rat ADMSCs, induced by dipsacus saponin VI(10μmol / L) for 15 days, were observed under the inverted microscope. Calcific knobs and osteocalcin levels were detected by Alizarin Red staining and immunocytochemistry, ELISA respectively. The artificial bone grafts, formed by the co-culture of ADMSCs and graft, were co-induced by dipsacus saponin VI for 15 days. The survival statement of cells on the artificial bone grafts were detected by trypan blue staining at 3d,6d,9d,12 d and 15 d respectively.Bone defect experiment were divided into 4 groups as follow: ⑴blank control, ⑵ADMSCs group(cell group), ⑶ADMSCs and n-HA / CS composite group(binary complex group) and ⑷ADMSCs and n-HA / CS / TSF composite group(ternary complex group) at random. The rats models were sacrificed post-operation 4, 8 and 12 weeks respectively. The X-ray film, for the bone defect regions, were analyzed according to Gary standard.The radius, contained bone defect, were separated and viewed under naked eyes. The new bone formation, detected by the routine HE staining, were evaluated according to Nilsson’s histological standard.The all results were statistical analysis by SPSS13.0 statistical software.ResultsThe primary ADMSCs, adherent growth, were in relatively uniform spindle cells and parallel pattern growth. CD44, mesenchymal stem cell-related markers was high expression. proliferative capacity of ADMSCs were not significantly reduced after repeated subculture by MTT detection. The phenotype of induced cells were triangle, square or polygonal shape and connected each other in some place. The red particles, formed by the calcium deposits, were found by Alizarin Red staining. The positive ratio of osteocalcin expression were 85 ± 5.22% detected by Immunocytochemistry methods. After induced 9th days, the expression level of osteocalcin were increased accompany with the extension of the induction time by ELISA.The alive cells on the graft, co-cultured of ADMSCs and graft, were increased accompany extension of induction time.The bone defect region were only contained some lower density shadow of blank control group and cell group under the X-ray postoperation 4 weeks. The blunt ends and the enclosed marrow cavity were also found basing on the X-ray film. The new bones were not clearly. The artificial grafts of binary and ternary material group were partially absorbed. And the contents of the ternary group were absorbed more than that of binary group. Partial compact bone of two ends of bone defect were connected each other in binary and ternary groups post-operative weeks. And the high density callus could be found in these two groups. The new formed bone was connected the two ends of bone defect in ternary group and its diameter was thicker than that of the binary group postoperative 12 W. And many bone marrow cavities of bone defect were continuous. There was no significant statistics sense between the blank control and cell groups according to Gary scores(P>0.05). Gary scores of ternary group were higher than that of binary group or cell group and there were significant statistics sense between the ternary group and binary group or cell group(P<0.05).There were no bone tissue-like structures in blank control and cell groups. The bone defect were only filled with fibrous tissue at each time points. The artificial graft were mainly absorbed in ternary group at 4 weeks and the spaces in or out of the graft were fully filled by cells. The artificial graft were partially absorbed in binary group and the cells could be seen around the graft. The artificial graft were all degradation and new capillaries were grown could be found in graft place in binary and ternary groups post-operation 8 weeks. There were more capillaries and new bone units-liked structures in the bone defect region at 12 weeks. The bone matrix color, in pale pink, was lower than that of normal bone tissue, indicating that the matrix is not yet mature.Nilsson scores of ternary group were significant higher than that of binary and cell groups post-operation 4, 8 and 12 weeks(P <0.05).ConclusionThe rat adipose-derived mesenchymal stem cells could be differentiated into osteoblasts induced by Dipsacus saponin Ⅵ. The artificial bone graft, formed by induced ADMSCs and n-HA / CS / TSF ternary vector, was a better graft for bone defect reparation. |
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